Segmental bone defects and nonunions are relatively common in the craniofacial skeleton. Osteogenic proteins, including bone morphogenetic protein-2 and 4 (BMP-2,4), can promote bone healing in segmental bone defects, but the osteogenic proteins' short half-lives and rapid clearance by the bloodstream limit the success of this technology. Gene therapy and tissue engineering approaches that can achieve high expression levels of these proteins may help to further improve craniofacial bone healing. Our recently isolated clonal population of muscle-derived stem cells (mcl3 cells) that can express stem cell markers, differentiate into both myogenic and osteogenic lineages, and, more importantly, improve bone healing in a calvarial bone defect may be an ideal cell population to mediate gene transfer of osteogenic proteins. The long-term goal of this proposed project is the development of gene therapy approaches based on this novel population of muscle-derived stem cells to efficiently deliver the osteogenic proteins and improve craniofacial bone healing. The mechanism by which these muscle-derived stem cells differentiate into osteogenic lineages under the influence of BMP-2 and BMP-4 will be tested and compared. In addition, we propose to characterize designated approaches of muscle-based tissue engineering using the muscle-derived stem cells in an ex vivo gene transfer of osteogenic proteins in combination with a scaffold to improve bone healing in a mouse calvarial defect. We will investigate the persistence of osteogenic protein expression, the presence of immune response and undesirable side effects related to the overexpression of these proteins, and the biological effects on fracture healing. The use of vascular endothelial growth factor (VEGF), a well-known angiogenic factor, to further improve bone healing will be also characterized. Although this proposed research will focus on muscle-based tissue engineering to regenerate a calvarial defect, this technology ultimately will be applied to other craniofacial sites, as well as appendicular bones. The proposed research will enhance and expand our knowledge of bone healing and develop a clinically relevant treatment based on new molecular therapeutics to treat osseous deficiencies.